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Abstract

Introduction

The aim was to investigate the prevalence of endotoxemia in children admitted to pediatric
intensive care unit (PICU), and its association with disease severity and outcome.

Methods

We conducted a prospective, observational cohort study of children admitted to PICU
at St. Mary's Hospital, London over a 6-month period. One hundred consecutive patients
were recruited. Demographic and clinical data were collected. Severity of illness
was assessed by the pediatric index of mortality 2 (PIM2) score. The pediatric logistic
organ dysfunction (PELOD) score was performed daily for the first 4 days. Patients
were categorized according to primary reason for PICU admission. Blood samples were
taken within 24 hours of admission and endotoxemia was measured using the endotoxin
activity assay (EAA). Patients were stratified according to EAA level (high, EAA >
0.4, low, EAA < 0.4) and categorized as septic, post-surgical, respiratory or other.
Data were analyzed using appropriate non-parametric tests.

Results

EAA level was significantly lower in PICU controls versus other PICU admissions (P = 0.01). Fifty-five children had endotoxemia on admission. Forty-one (75%) of these
were eventually diagnosed with an infectious cause of admission. Nine children without
infection had elevated EAA on admission. An infectious cause of admission was significantly
associated with endotoxemia (P < 0.005). Of 15 children with gram-negative infection, only 9 (60%) had endotoxemia
on admission. Endotoxemia on admission was not associated with shock or death. However,
there was a tendency for increased PELOD score and length of stay in endotoxemic children.

Conclusions

Endotoxemia is common in children admitted to intensive care. Understanding the implications
of endotoxemia and potential anti-endotoxin strategies may have the potential to reduce
severity of illness and length of PICU stay in critically ill children.

Introduction

Sepsis is a major cause of admission to pediatric intensive care units (PICUs) and
causes significant morbidity and mortality in children. A recent study from the US
estimated that the incidence of pediatric severe sepsis is 0.56 cases per 1,000 population
and that severe sepsis has an overall hospital mortality rate of 10.3% and accounts
for 7% of all deaths in children [1].

In 2009, there were nearly 17,000 admissions to PICUs in the UK. Of these, nearly
9,000 (53%) were unplanned medical admissions for conditions such as sepsis, pneumonia,
bronchiolitis, and respiratory failure (Pediatric Intensive Care Audit Network [PICANET]
Annual Report 2009) [2]. The unadjusted case fatality rate for children admitted to PICUs in the UK is 4.1%
and these children account for over 100,000 bed days. A recent audit of referrals
of children with sepsis to PICUs in the UK found that 17% of these children died [3].

The earliest events in the pathogenesis of sepsis are interactions of pathogen-related
antigens (for example, endotoxin (lipopolysaccharide, LPS) and peptidoglycan) with
cell surface pattern recognition receptors such as the Toll-like receptors 4 and 2,
respectively [4]. The interaction of LPS with its receptors and the subsequent cellular responses
have been well described and are pivotal processes in the inflammatory response leading
to the manifestations of sepsis [5].

Measuring the concentration of LPS in human disease has been notoriously difficult.
The most commonly used method, the chromogenic limulus amebocyte lysate (LAL) assay,
is based on the ability of endotoxin to induce coagulation of hemolymph in the horseshoe
crab, Limulus polyphemus [6]. The utility of this assay has been limited because of circulating inhibitors of
the coagulation reaction. In addition, the assay is not specific for endotoxin.

A novel endotoxin assay, which is simpler and more accurate than the LAL assay, was
recently described [7]. This endotoxin activity assay (EAA) detects LPS in whole blood by the use of neutrophil-dependent
chemiluminescence. This assay was used in a study of critically ill adults, in which
an association between endotoxemia with infection and an increased risk of adverse
outcome was demonstrated [8]. Because of the link between endotoxin and inflammation, we sought to define the
prevalence of endotoxemia in critically ill children and determine the association
of endotoxemia with infection, severity of illness, and outcome.

Materials and methods

We undertook a prospective observational cohort study of critically ill children admitted
to the PICU at St Mary's Hospital, London, over the course of a 6-month period (January
to June 2007). The unit does not admit cardiac-surgical or neurosurgical patients.
Informed consent was obtained from parents or carers. The study was approved by the
local research ethics committee.

We defined a PICU control group as children who were electively admitted to the PICU
for post-operative care. They were all electively ventilated but had no other organ
failure on admission.

Conditions were diagnosed as sepsis or septic shock (or both) according to the criteria
of Goldstein and colleagues [9]. Infection was diagnosed with standard microbiological techniques.

Severity of illness was assessed by the pediatric index of mortality 2 (PIM2) score
[10], and the pediatric logistic organ dysfunction (PELOD) score [11] was performed daily for the first 4 days (as 4 days was the mean length of stay,
or LOS).

Chemiluminescent assay for endotoxin

All patients had a single measurement of endotoxin activity (EA) in whole blood within
24 hours of PICU admission, as described previously [7]. Whole blood samples (2 mL) were collected into ethylenediaminetetraacetic acid (EDTA)
vacutainer tubes. Samples of 0.5 mL of whole blood in duplicate were immediately incubated
with saturating concentrations of a murine IgM monoclonal antibody against the lipid
A of Escherichia coli J5. This antibody is broadly cross-reactive against Gram-negative bacteria but does
not cross-react with Gram-positive bacteria. Any LPS present in the blood complexes
with the anti-LPS antibody. This complex primes the patient's neutrophils for an augmented
response to stimulation with zymosan. The resulting respiratory burst activity is
detected as light release from the lumiphor luminol by using a chemiluminometer (Autolumat
LB953; EG&G Berthold, Bad Wildbad/Germany). By measuring basal (no antibody) and maximally
stimulated (4,600 pg/mL LPS) responses in the same blood sample, the EA of the test
specimen is calculated by integrating chemiluminescence over time. Levels are expressed
as EA units and represent the mean of duplicate determinations from the same sample.
An EA level of greater than 0.4 is approximately equivalent to an endotoxin concentration
of 25 to 50 pg/mL E. coli 055:B5 LPS.

As the EAA requires adequate numbers of patient neutrophils to provide the respiratory
burst for the assay readout, we excluded patients who had an absolute neutropenia
of less than 0.1 × 106/cm3. For the purposes of this study, a cutoff level of 0.4 EA units, as defined previously
[7], was used to determine the presence of endotoxemia (that is, ≤0.4 negative, > 0.4
EA present).

Statistics

Data were non-parametric and are presented as median ± interquartile range (IQR).
The results were analyzed by using GraphPad Prism (GraphPad Software, Inc., La Jolla,
CA, USA). The Mann-Whitney U test was used to compare groups of data, and the Kruskal-Wallis test was used if there
were more than two groups of data. The chi-square test was used to compare proportions.

Results

One hundred four consecutive admissions were asked to participate in the study but
four of them refused. No children were excluded; therefore, 100 patients were recruited
for the study. Their demographics are shown in Table 1. Ten children (10%) died and 90 were discharged alive from the PICU. The mean LOS
for survivors was 7.1 days (median of 5 days and range of 1 to 57). There were 9 children
in the control group, 48 in the respiratory group, 18 in the sepsis group, and 25
in the 'other' group. The control group consisted of two children who had tonsillectomy
and adenoidectomy for obstructive sleep apnea, three children who had a Nissen fundoplication
and gastrostomy, one child who had a gastric pull-up for esophageal atresia, one child
who had an esophageal dilatation, one child who had a resection of a colonic duplication
cyst, and one child who had a partial nephrectomy. None suffered periods of intra-operative
hypotension or needed prolonged post-operative ventilation.

Fifty-five children had endotoxemia on admission to the PICU. Only two of these were
in the control group (one child underwent an elective Nissen fundoplication and had
extensive bowel manipulation during surgery, and the other child underwent an adenotonsillectomy).
Of these 55 children, 41 (75%) were eventually given a diagnosis of an infection-related
cause of admission (Table 2).

Seventeen children were bacteremic on admission. Fifteen children had Gram-negative
infection as the cause of admission, and only nine (60%) of them were endotoxemic
on admission. Of the 15 children with Gram-negative infection, only 5 had Gram-negative
bacteremia (all meningococcus serogroup B). Only 3 of the 5 children with meningococcal
bacteremia had endotoxemia detected on admission.

Sixty-two children were given a diagnosis of an infection-related cause of admission,
and 41 (66%) of them had endotoxemia and 14 of 38 children (37%) had a non-infectious
cause of admission (P = 0.0043, chi-square test). Seventy-five percent of children with respiratory failure
of any cause had endotoxemia on admission to the PICU.

All children admitted to our PICU with a suspicion of infection are treated with a
standard regimen of anti-microbials, consisting of a third-generation cephalosporin
(usually ceftriaxone) with or without gentamicin if there is a suspicion of pseudomonas
infection (such as patients with chronic lung disease). There was no clear correlation
between anti-microbial use and endotoxemia (data not shown).

The presence of endotoxemia on admission to the PICU was not significantly associated
with shock or death. Twenty-two of 34 children (64%) who had shock at PICU admission
had endotoxemia compared with 35 out of 66 (53%) without shock (P = not significant, chi-square test). Seven of the 10 children (70%) who died had endotoxemia
on admission compared with 48 out of 90 survivors (53%) (P = not significant, chi-square test). Daily PELOD score, PIM2 score on admission, and
LOS in survivors were not significantly different between the groups with and without
endotoxemia on admission, although there was a tendency for increased PELOD score
and LOS in the group with endotoxemia (Table 3).

Discussion

Our study shows that endotoxemia is common in a heterogeneous population of critically
ill children. Although there was no clear correlation with severity of illness or
the presence of shock, there was a tendency toward longer LOS in patients with endotoxemia.
This is consistent with a recent study in adult surgical intensive care patients,
which showed an association between endotoxemia and LOS in the intensive care unit
[12].

Despite the high prevalence of endotoxemia found in our study, only 15 children had
a Gram-negative infection, a scenario classically associated with endotoxemia. In
addition, only 9 of 15 children with documented Gram-negative infection had endotoxemia.
This may reflect the timing of the assay in relation to admission and treatment. It
has previously been noted that endotoxin level in blood declines rapidly following
appropriate anti-microbial therapy [13]. We did not record the timing of performance of the EAA in relation to the start
of anti-microbial therapy, but 75% of the samples were taken within 12 hours of admission
to the PICU.

The commonest cause of PICU admission in our study was respiratory failure that was
usually due to viral lower respiratory tract infection. The majority (75%) of children
with a respiratory tract infection had endotoxemia on admission despite having low
severity-of-illness scores and good outcome. Several studies have documented high
levels of endotoxin in the lung and blood of patients with respiratory infection as
well as those on artificial ventilation. Pulmonary-to-systemic translocation of LPS
has been demonstrated in non-protective ventilation strategy studies in experimental
animals [14], and an association between ventilator-induced lung injury, ventilator-associated
pneumonia, and systemic LPS and cytokine levels has been reported [15]. Although the lung has no endogenous microflora that could provide a source of LPS,
it is likely that bacterial colonization of the airway or lung may be a potential
source of LPS that can translocate systemically following mechanical ventilation.
The high prevalence of endotoxemia in our patients seems to confirm this.

The gut provides a second and major potential source of systemic LPS. The indigenous
flora of the gastrointestinal tract contains large amounts of endotoxin, and translocation
of both endotoxin and viable bacteria from the gut has been demonstrated in animal
models and in human illness associated with splanchnic hypoperfusion [16,17]. Consistent with this, we observed that endotoxemia was more common in critical illness
compared with control children. In addition, patients who had endotoxemia on admission
appeared to be a sicker population, as reflected by longer LOS and a trend toward
higher PELOD scores. Thus, the presence of endotoxemia on admission may identify a
high-risk subpopulation of critically ill children.

The chemiluminescent assay used in our study is both sensitive and specific for endotoxin
and can be performed in less than 1 hour, permitting the rapid detection of endotoxin
in fresh whole blood. However, it uses the patient's own neutrophils as a readout
system and so presents inherent limitations; in particular, it is not possible to
store specimens for later assay; measurements must be performed within 3 hours of
obtaining the sample. Because of the requirement for neutrophils, the assay is not
reliable in patients with an absolute neutrophil count of less than 0.1 × 106/cm3. The assay relies on neutrophil chemiluminescence in response to IgM anti-LPS antibody
and LPS. However, the pre-activation state is accounted for by correcting for baseline
chemiluminescence in the EAA. In addition, the assay detects exposed lipid A in the
endotoxin molecule and so may not reflect endotoxin bioactivity in vivo. However, the availability of a relatively simple bedside assay for blood endotoxin
level may identify a high-risk population of critically ill patients who may benefit
from adjunctive therapy.

We did not correlate the EAA with the best-known endotoxin assay, the LAL assay. The
LAL assay has been widely used to detect endotoxin contamination of drugs and fluids;
however, its utility in biological samples has been limited because of circulating
inhibitors of the coagulation reaction. In addition, other microbial products, notably
from fungi, can activate the LAL reaction, so the assay is not specific for endotoxin
[18]. Studies comparing EAA and LAL show considerable variability in the prevalence of
endotoxemia or its association with Gram-negative infection, and the EAA is able to
detect endotoxemia associated with Gram-negative infection from any source, a diagnosis
of sepsis, and an elevated white blood cell count; no such correlations were found
when samples were assayed using the LAL method [19].

Antibiotics can accelerate endotoxin release and may result in false-negative blood
cultures [20]. We did not correlate endotoxemia with type and duration of anti-microbial therapy.
However, most patients would have been treated with our typical empiric anti-microbial
regimen, which consisted of a third-generation cephalosporin, with additional gentamicin
in certain cases, such as those with specific risk factors for pseudomonas infection.

Whether the increased LOS and possible increased severity of illness associated with
endotoxemia might be reduced by specific measures to neutralize endotoxin needs to
be studied further, but the hypothesis remains attractive. High levels of endotoxin
have been associated with increased severity of illness in meningococcal disease [13], and studies of anti-endotoxin therapy in human sepsis suggest that the greatest
potential benefit occurs in patients in whom endotoxemia is likely to be present [21].

Inferences from the present study are limited by its observational nature and the
relatively small number of patients in each disease category. In addition, the timing
of the assay may have 'missed' the peak of endotoxemia in those patients in whom anti-microbial
therapy had been initiated prior to PICU admission. In addition, intrinsic anti-EA
(such as endotoxin antibodies) and lipoproteins may quench EA.

Conclusions

The demonstration of significant endotoxemia in a high proportion of children admitted
to the PICU for any one of several pathologies suggests an important interaction between
critical illness and inflammation induced or augmented by intrinsically derived endotoxin.
It is possible that this endotoxemia contributes to severity of illness and outcome.
The hypothesis that LPS contributes to disease severity in critical illness requires
further exploration. Our study suggests that endotoxemia in critically ill children
is common and can be detected with a simple bedside test. The use of anti-endotoxin
agents potentially may have a role in the treatment of critical illness of all causes
in children, and this should be the subject of future studies.

Key messages

• Children with critical illness of any cause are likely to have endotoxemia.

• Endotoxemia can be accurately detected by a simple bedside test.

• Endotoxemia may be associated with increased severity of illness and length of stay
in the pediatric intensive care unit.

• It is likely that the source of endotoxin in these children is their own gut or
lung.

• If the findings of this study are confirmed in larger studies, anti-endotoxin therapies
may be a logical adjunct to the treatment of pediatric critical illness.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

SD recruited patients, performed the assay, and wrote the first draft of the manuscript.
DI gave statistical advice and contributed to the manuscript. HB recruited patients,
performed the assay, and contributed to the manuscript. SN initiated the project,
recruited patients, performed the assay, and contributed to the manuscript. All authors
read and approved the final manuscript.

Acknowledgements

SN and DI are grateful for support from the NIHR Biomedical Research Centre funding
scheme. The funding agencies had no role in study design, data management, analysis,
or manuscript preparation. We would like to acknowledge the following, without whom
this study would not have been possible: David Klein and Debra Foster of Spectral
Diagnostics Inc. (Toronto, ON, Canada), who provided technical advice and supported
use of the EAA; COSMIC (Children of St Mary's Intensive Care) medical charity, which
provided financial support; the nursing staff from the PICU at St Mary's, for coordination
and patient recruitment; and the children involved in the study and their parents.

References

Watson RS, Carcillo JA, Linde-Zwirble WT, Clermont G, Lidicker J, Angus DC: The epidemiology of severe sepsis in children in the United States.